Method for determining the ability of an antibody to keep cells close to one another

Abstract

The invention relates to a method for determining the ability of a candidate antibody to keep a first cell and a second cell close to one another, said method comprising the following steps: bringing said candidate antibody in contact with (i) a first cell expressing, in the extracellular portion of its plasma membrane, a first protein that is known to be or is suspected of being recognized by the candidate antibody and (ii) a second cell expressing, in the extracellular portion of the plasma membrane, a second protein that is also known to be or suspected of being recognized by the candidate antibody, each of the proteins being labeled with a member of a pair of FRET partners, and measurement of the FRET signal and comparison with the signal measured in the absence of the candidate antibody. The invention also relates to a reagent kit for carrying out this method.

1 . A method for determining the ability of a candidate antibody to keep a first cell and a second cell close to one another, this method comprising the following steps:
 bringing the following elements in contact:
 (i) a first cell expressing, in the extracellular portion of its plasma membrane, a first protein that is known to be or is suspected of being recognized by the candidate antibody, said first protein being labeled directly or indirectly with the first member of a pair of FRET partners, 
 (ii) a second cell expressing, in the extracellular portion of the plasma membrane, a second protein that is also known to be or suspected of being recognized by the candidate antibody, said second protein being labeled directly or indirectly with the second member of said pair of FRET partners, 
 (iii) said candidate antibody; 
   measuring of the FRET signal and comparison with the signal measured in the absence of the candidate antibody, wherein an increase in the signal measured in the presence of the candidate antibody relative to that measured in its absence is indicative of the ability of said antibody to keep the first cell and the second cell close to one another.   
     
     
         2 . The method as claimed in  claim 1 , wherein said first protein is a membrane receptor of the Fc fragment of the antibodies, and wherein said second protein bears an epitope recognized by at least one of the paratopes of the candidate antibody. 
     
     
         3 . The method as claimed in  claim 2 , wherein the Fc receptor is an Fc gamma receptor. 
     
     
         4 . The method as claimed in  claim 2 , wherein the Fc receptor is the CD16a receptor or a variant thereof. 
     
     
         5 . The method as claimed in  claim 1 , wherein the candidate antibody comprises two different paratopes, the first paratope being specific for a first epitope and the second paratope being specific for a second epitope, in that said first protein bears said first epitope, and wherein said second protein bears said second epitope. 
     
     
         6 . The method as claimed in  claim 5 , wherein said first protein is selected from: CD3, CD28. 
     
     
         7 - 8 . (canceled) 
     
     
         9 . The method as claimed in  claim 1 , wherein either the first protein, or the second protein, or the first and the second proteins are labeled directly via a suicide enzyme, and wherein the labeling is carried out by bringing the cells expressing these proteins into contact with the substrate of the enzyme, conjugated to one member of the pair of FRET partners. 
     
     
         10 . The method as claimed in  claim 9 , wherein the suicide enzyme is selected from: the mutants of O6-alkylguanine DNA alkyltransferase, the mutants of dehalogenase, and a fragment of the acyl carrier protein. 
     
     
         11 . A reagent kit for carrying out the method as claimed in  claim 1 , which comprises the following components:
 mammalian cells expressing a first membrane protein fused to a suicide enzyme;   mammalian cells expressing a second membrane protein fused to a suicide enzyme;   the substrates of the suicide enzymes, conjugated to the members of a pair of FRET partners.   
     
     
         12 . A reagent kit for carrying out the method as claimed in  claim 1 , which comprises the following components:
 mammalian cells expressing a first membrane protein fused to a suicide enzyme and labeled with the first member of a pair of FRET partners;   mammalian cells expressing a second membrane protein fused to a suicide enzyme and labeled with the second member of a pair of FRET partners.   
     
     
         13 . The method as claimed in  claim 3 , wherein the Fc receptor is the CD16a receptor or a variant thereof. 
     
     
         14 . A method for determining the cytotoxic character of a candidate antibody, which method comprises the steps of:
 bringing the following elements in contact:
 (i) a first cell expressing, in the extracellular portion of its plasma membrane, a first protein that is known to be or is suspected of being recognized by the candidate antibody, said first protein being labeled directly or indirectly with the first member of a pair of FRET partners, wherein said first cell is a cell imitating an effector cell selected from: NK lymphocytes, macrophages, cytotoxic T lymphocytes, and helper T cells; 
 (ii) a second cell expressing, in the extracellular portion of the plasma membrane, a second protein that is also known to be or suspected of being recognized by the candidate antibody, said second protein being labeled directly or indirectly with the second member of said pair of FRET partners; 
 (iii) said candidate antibody; 
   measuring the FRET signal, wherein the appearance of a FRET signal is representative of the affinities of the candidate antibody for the first and the second cells.   
     
     
         15 . The method as claimed in  claim 14 , wherein said first protein is a membrane receptor of the Fc fragment of the antibodies, and wherein said second protein bears an epitope recognized by at least one of the paratopes of the candidate antibody. 
     
     
         16 . The method as claimed in  claim 15 , wherein the Fc receptor is an Fc gamma receptor. 
     
     
         17 . The method as claimed in  claim 14 , wherein the candidate antibody comprises two different paratopes, the first paratope being specific for a first epitope and the second paratope being specific for a second epitope, wherein said first protein bears said first epitope, and wherein said second protein bears said second epitope. 
     
     
         18 . A method for determining the cytotoxic character of a candidate antibody, which method comprises the steps of:
 bringing the following elements in contact:
 (iv) a first cell expressing, in the extracellular portion of its plasma membrane, a first protein that is known to be or is suspected of being recognized by the candidate antibody, said first protein being labeled directly or indirectly with the first member of a pair of FRET partners, wherein said first protein is selected from: CD16a, CD3, and CD28; 
 (v) a second cell expressing, in the extracellular portion of the plasma membrane, a second protein that is also known to be or suspected of being recognized by the candidate antibody, said second protein being labeled directly or indirectly with the second member of said pair of FRET partners; 
 (vi) said candidate antibody; 
   measuring the FRET signal, wherein the appearance of a FRET signal is representative of the affinities of the candidate antibody for the first and the second cells.   
     
     
         19 . The method as claimed in  claim 18 , wherein said first protein is CD16a.